This application is a reissue application of U.S. application Ser. No. 08/612,857, filed Mar. 13, 1996, which is a xc2xa7371 national stage filing of PCT/FR94/00542, filed May 9, 1994.
The present invention relates to a new gene, designated Grb3-3, its variants, and their uses, especially in anti-cancer gene therapy.
Various genes, called oncogenes and suppressor genes, are involved in the control of cell division. Among them, the ras genes and their products generally designated p21 proteins, play a key role in the control of cell proliferation in all eukaryotic organisms where they have been searched out. In particular, it has been shown that certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumours have been associated with the presence of modified ras genes. Likewise, an overexpression of these p21 proteins can lead to a deregulation of cell proliferation. An understanding of the exact role of these p21 proteins in cells, of their mode of operation and their characteristics therefore constitutes a major stake for the understanding and the therapeutic approach to carcinogenesis.
Various factors involved in the ras-dependent signalling pathway have been identified. Among these are the Grb2 gene which encodes a protein of 23-25 kDa having a SH3-SH2-SH3 structure (Lowenstein et al., Cell 70 (1992) 431; Matuoka et al., PNAS 89 (1992) 9015). The product of the Grb2 gene appears to interact with the tyrosine phosphorylated proteins via its SH2 domain, and with a factor for exchange of GDP of the SOS class via its SH3 domain (Egan et al., Nature 363 (1993) 45). It would thus be one of the components of the transformant activity of the product of the ras gene. The present invention derives from the demonstration of the cloning and characterization of an isoform of the Grb2 gene, designated Grb3-3, possessing a deletion in the SH2 domain. This gene is expressed in adult tissues: the corresponding mRNA is present in the form of a single band of 1.5 kb, and is translated into a 19 kDa protein. Because of its deletion in the SH2 domain, the product of the Grb3-3 gene is no longer capable of interacting with the tyrosine phosphorylated proteins (phosphorylated EGF receptor), but it retains the capacity to interact with the proline-rich domains of the SOS proteins. Because of its deletion, the product of the Grb3-3 gene is thus capable of preventing the cellular effects of the product of the Grb2 gene. The transfer of this gene in vivo, or of variants thereof, including antisense sequences, therefore makes it possible to interfere with the processes of proliferation, differentiation and/or cell death.
A first subject of the invention therefore relates to a nucleotide sequence comprising all or part of the Grb3-3 gene (sequence SEQ ID No. 1).
Another subject of the invention relates to a nucleotide sequence derived from the sequence SEQ ID No. 1 and capable of inhibiting, at least partially, the expression of the Grb2 or Grb3-3 protein. In particular, the invention relates to the antisense sequences whose expression in a target cell makes it possible to control the transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNAs complementary to the cellular mRNAs Grb2 or Grb3-3 and thus block their translation into protein, according to the technique described in patent EP 140 308. Such sequences may consist of all or part of the nucleic sequence SEQ ID No. 1, transcribed in the reverse orientation.
As indicated above, Grb2 is a protein which is at least bifunctional, and which is anchored via its SH2 domain to specific sequences phosphorylated at the tyrosine, and via its two SH3 domains, to the exchange factors of the SOS family. Grb3-3 having lost its capacity to associate with proteins phosphorylated at the tyrosine can therefore only form a complex with the SOS proteins. Grb3-3 can therefore prevent the recruitment of the Grb2-SOS complex by the receptors of the self-phosphorylated growth factors or by associated proteins which are also phosphorylated at the tyrosine such as HSC or IRS1. Grb3-3 being capable of blocking this recruitment, it is capable of blocking mytogenic pathways and of inducing cell death. The Applicant has indeed demonstrated that the Grb3-3 protein was expressed during certain physiological processes such as for example the maturation of the thymus in rats. The Applicant has also shown that Grb3-3 is capable of inducing cell death by apoptosis of various cell types. It was possible to detect these completely advantageous properties (i) by injecting recombinant protein into the 3T3 fibroblasts and (ii) by transferring the sequence encoding Grb3-3 into the 3T3 cells (Example 4). Grb3-3 is therefore capable of inducing the cellular death of viable cells such as immortalized, cancer or embryonic cells. As shown in the examples, Grb2 is capable of preventing the effects of Grb3-3.
Moreover, a search for the expression of Grb3-3 carried out during the infection of lymphocytic cells by the HIV virus made it possible to show that the massive viral production observed 7 days after the infection is correlated with an overexpression of the Grb3-3 mRNA by the infected cells (Example 5). This experiment shows that eliminating or blocking the cellular effects of Grb3-3 can also make it possible to maintain alive cells infected especially with HIV, and thus allow the T4 lymphocytes to continue to play a role of immune defence. In this respect, the invention also relates to the use of compounds capable of eliminating or blocking, at least partially, the cellular effects of Grb3-3 for the preparation of a pharmaceutical composition intended for the treatment of AIDS. More particularly, the compounds used may be:
genetic antisense sequences such as those defined above,
oligonucleotides specific to Grb3-3, modified or otherwise for better stability or bioavailability (phosphorothioates, intercalating agents and the like). They may be preferably oligonucleotides covering the coding sequence localized between the N-terminal SH3 domain and the residual SH2 domain,
any sequence whose transfer into the infected cells induces an overexpression of Grb2.
The nucleic acid sequences according to the invention can be used as such, for example after injection into man or animals, to induce a protection or to treat cancers. In particular, they can be injected in the form of naked DNA according to the technique described in application WO 90/11092. They can also be administered in complexed form, for example with DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), with nuclear proteins (Kaneda et al., Science 243 (1989) 375), with lipids (Felgner et al., PNAS 84 (1987) 7413), in the form of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like.
Preferably, the nucleic acid sequences according to the invention form part of a vector. The use of such a vector indeed makes it possible to improve the administration of the nucleic acid into the cells to be treated, and also to increase its stability in the said cells, which makes it possible to obtain a durable therapeutic effect. Furthermore, it is possible to introduce several nucleic acid sequences into the same vector, which also increases the efficacy of the treatment.
The vector used may be of diverse origin, as long as it is capable of transforming animal cells, preferably human tumour cells. In a preferred embodiment of the invention, a viral vector is used which can be chosen from adenoviruses, retroviruses, adeno-associated viruses (AAV), herpes virus, cytomegalovirus (CMV), vaccinia virus and the like. Vectors derived from adenoviruses, retroviruses or AAVs incorporating heterologous nucleic acid sequences have been described in the literature [Akli et al., Nature Genetics 3 (1993) 224; Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185 573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al., Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; WO91/18088].
The present invention therefore also relates to any recombinant virus comprising, inserted into its genome, a nucleic acid sequence as defined before.
Advantageously, the recombinant virus according to the invention is a defective virus. The term xe2x80x9cdefective virusxe2x80x9d designates a virus incapable of replicating in the target cell. Generally, the genome of the defective viruses used within the framework of the present invention is therefore devoid of at least the sequences necessary for the replication of the said virus in the infected cell. These regions can either be removed (completely or partially), or rendered non-functional, or substituted by other sequences and especially by the nucleic acid of the invention. Preferably, the defective virus nevertheless conserves the sequences of its genome which are necessary for the encapsulation of the viral particles.
It is particularly advantageous to use the nucleic acid sequences of the invention in a form incorporated in an adenovirus, an AAV or a defective recombinant retrovirus.
As regards adenoviruses, various serotypes exist whose structure and properties vary somewhat, but which are not pathogenic for man, and especially non-immunosuppressed individuals. Moreover, these viruses do not integrate into the genome of the cells which they infect, and can incorporate large fragments of exogenous DNA. Among the various serotypes, the use of the type 2 or 5 adenoviruses (Ad2 or Ad5) is preferred within the framework of the present invention. In the case of the Ad5 adenoviruses, the sequences necessary for the replication are the E1A and E1B regions.
The defective recombinant viruses of the invention can be prepared by homologous recombination between a defective virus and a plasmid carrying, inter alia, the nucleotide sequence as defined above (Levrero et al., Gene 101 (1991) 195; Graham, EMBO J. 3(12)(1984) 2917). The homologous recombination is produced after co-transfection of the said viruses and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by the said elements, and (ii), contain sequences capable of complementing the part of the genome of the defective virus, preferably in integrated form so as to avoid the risks of recombination. As example of a line which can be used for the preparation of defective recombinant adenoviruses, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains especially, integrated into its genome, the left part of the genome of an Ad5 adenovirus (12%). As example of a line which can be used for the preparation of defective recombinant retroviruses, there may be mentioned the CRIP line (Danos and Mulligan, PNAS 85 (1988) 6460).
Then the viruses which have multiplied are recovered and purified according to conventional molecular biology techniques.
The subject of the present invention is also a pharmaceutical composition containing at last one recombinant virus or a nucleotide sequence as defined above.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected, optionally directly into the tumour to be treated. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The doses of nucleic acids (sequence or vector) used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, of the nucleic acid to be expressed, or alternatively of the desired duration of treatment. Generally, with regard to the recombinant viruses according to the invention, the latter are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, and preferably 106 to 1010 pfu/ml. The term pfu (xe2x80x9cplaque forming unitxe2x80x9d) corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate cell culture and measuring, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.
Such pharmaceutical compositions can be used in man, for the treatment and/or prevention of cancer. In particular the products of the invention are capable of modulating the activity of ras proteins, they make it possible to intervene in the cancer development process, and in particular, they can inhibit the activity of oncogenes whose transformant activity depends on a p21-functional GAP interaction. Numerous cancers have indeed been associated with the presence of oncogenic ras proteins. Among the cancers most often containing mutated ras genes, there may be mentioned especially adenocarcinomas of the pancreas, of which 90% have a Ki-ras oncogene mutated on the twelfth codon (Almoguera et coll., Cell 53 (1988) 549), adenocarcinomas of the colon and cancers of the thyroid (50%), or carcinomas of the lung and myeloid leukaemias (30%, Bos, J. L. Cancer Res. 49 (1989) 4682). More generally, the compositions according to the invention can be used for treating any type of pathology in which an abnormal cell proliferation is observed, by inducing apoptosis, as well as any pathology characterized by a cell death by apoptosis (AIDS, Huntington""s chorea, Parkinson), by means of compounds which block the effects of Grb3-3 (antisense in particular).
The present invention will be more fully described with the aid of the following examples which should be considered as illustrative and non-limiting.